Tuberous sclerosis complex (TSC) is an autosomal disorder resulting from mutations in the TSC1 or TSC2 genes that is associated with epilepsy, cognitive disability, and autism. TSC1/TSC2 gene mutations lead to developmental alterations in brain structure known as tubers in over 80% of TSC patients. Loss of TSC1 or TSC2 function in tubers results from biallelic TSC gene inactivation and leads to activation of the mTOR cascade as evidenced by phosphorylation of ribosomal S6 protein (P-S6). Several new findings warrant further investigation of the mechanisms through which TSC gene mutations lead to developmental alterations in brain structure. Recent MRI studies suggest that there are subtle widespread abnormalites in TSC brains that contribute to neurocognitive deficits and in vitro evidence suggests that reduction of Tsc1 in rat neurons leads to altered dendritic structure. First, we will define subtle structural alterations distinct from tubers in post-mortem TSC brain specimens in the cortex, thalamus, basal ganglia, and cerebellum which may contribute to neurocognitive abnormalites in TSC using neuronal and astrocytic protein markers. Then, in these non-tuber brain lesions we will define P- S6 expression as a strategy to determine whether cells in non-tuber brain lesions exhibit mTOR cascade activation similar to tubers. Next, we will identify somatic second hit mutations in single microdissected P-S6 labeled cells in non-tuber brain lesions as a strategy to define whether all structural abnormalites in TSC require biallelic TSC gene inactivation. Then, we will determine whether P-S6 labeled giant cells in tubers and non- tuber brain lesions express a single or multiple somatic second hit mutations to test the hypothesis that structural lesions form by a clonal cellular expansion. In the second experiments, we will transfect cultured rat neocortical neurons at embryonic day 16 with Tsc1 or Tsc2 shRNA to define the effects of reduced Tsc1 or Tsc2 on dendrite outgrowth and expression of dendritic mRNAs. In the third experiments, we will define the differential expression of microRNAs (miRNAs) in P-S6 labeled giant cells and in P-S6 labeled astrocytes from the Tsc1GFAP cre mouse strain. These short non-coding RNAs play a pivotal role in translational regulation and interact with several proteins including EIFs and STAT3 that mediate the effects of TSC1/TSC2 in neurons. These experiments provide new mechanistic strategies to define how loss of TSC1/TSC2 leads to altered brain structure is TSC. PUBLIC HEALTH RELEVANCE Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder that affects the brain, skin, kidney, heart, and lungs. The neurological manifestations include epilepsy, autism, hydrocephalus, and cognitive impairments. We propose to more fully define the extent of brain involvement in TSC by analyzing the brain structure of 10 post- mortem TSC brains with protein specific antibodies. In a second set of experiments we will define novel mechanisms that regulate dendrite outgrowth in neurons regulated by the TSC encoded proteins. These studies will provide new insights into the mechanisms leading to epilepsy, autism, and cognitive impairment in TSC.